Gamma-secretase is a fascinating multi-subunit intramembrane cleaving protease that is now being considered as a therapeutic target for a number of diseases. Primarily because gamma-secretase was a therapeutic target in Alzheimer's disease (AD), a number of GSIs have been developed that effectively inhibit gamma-secretase cleavage in humans. In humans with AD, long-term treatment with current GSIs appears to have mechanism based side-effects that limit the maximum tolerable dose to the point where the extent of long-term AB lowering is likely to be insufficient to have clinical impact in symptomatic patients. However, as elaborated on in Projects 1 and 2, because of its role in mediating signaling events from other proteins, with a primary focus on Notch signaling; gamma-secretase is a therapeutic target in cancer and immunologic disease and may very well be a target in other diseases, as long as the treatment window is acute, subacute or intermittent. Given the preliminary and published data in Projects 1 and 2 of this proposal as well as data from others supporting the repurposing of GSIs for multiple disease indications, further exploration of this ongoing repurposing effort is warranted. A major challenge when considering repurposing of GSIs is that the study of gamma-secretase cleavage has been dominated by its focus on APP and Notch 1. GSIs in AD are thought of as AB inhibitors and in cancer as Notch 1 inhibitors, but what about the GSI action on the nearly 100 additional substrates identified to date? Are these cleavages relevant or irrelevant to the biology of the system being investigated? Indeed, the major working hypothesis of this project is that in order to understand the biological role of gamma-secretase and appropriately repurpose GSIs for a given disease indication a fuller appreciation of gamma-secretase activity and the substrate inhibitory profiles of GSIs are needed. The studies proposed will enable facile assessment of cleavage of a broader repertoire of gamma-secretase substrates; guide the repurposing efforts for GSIs as therapeutics in cancer, GVHD and other emerging indications, and enable us to determine whether gamma-secretase cleavage is altered in aging or AD.